Identification and pathogenicity of Fusarium spp. associated with tea wilt in Zhejiang Province, China

Background Tea is one of the most widely consumed beverages in the world, with significant economic and cultural value. However, tea production faces many challenges due to various biotic and abiotic stresses, among which fungal diseases are particularly devastating. Results To understand the identity and pathogenicity of isolates recovered from tea plants with symptoms of wilt, phylogenetic analyses and pathogenicity assays were conducted. Isolates were characterized to the species level by sequencing the ITS, tef-1α, tub2 and rpb2 sequences and morphology. Four Fusarium species were identified: Fusarium fujikuroi, Fusarium solani, Fusarium oxysporum, and Fusarium concentricum. The pathogenicity of the Fusarium isolates was evaluated on 1-year-old tea plants, whereby F. fujikuroi OS3 and OS4 strains were found to be the most virulent on tea. Conclusions To the best of our knowledge, this is the first report of tea rot caused by F. fujikuroi in the world. This provides the foundation for the identification and control of wilt disease in tea plants.


Introduction
Tea (Camellia sinensis [L.] O. Kuntze) is a prominent plantation crop that belongs to the family Theaceae [1], which is cultivated in subtropical regions across Asia, Africa, and South America.As one of the most widely consumed beverages globally, tea holds significant importance in the global market [2].In 2022, China was the world's largest producer of tea, producing 2.8 million tonnes [3], with the majority of tea produced by smallholder farmers, who significantly benefited financially from the tea industry.
Despite its prominence, tea cultivation faces various biotic threats, and fungal diseases are the most serious, as they can cause significant losses in yield and quality.Among these diseases, blister blight, gray blight, brown blight, twig dieback, stem cankers, and root rots are considered to be the most severe [4].In particular, the genus Fusarium comprises soil-borne fungi known to cause wilt, leaf spot, collar canker, and dieback in tea plants [5,6].Fusarium collar canker and dieback are caused by Fusarium solani, which infects the stem and causes cracks, peeling of bark, white mold growth and eventual branch death in China and Sri Lanka [6,7].Fusarium proliferatum induces Fusarium leaf spot, characterized by the emergence of brown lesions with yellow halos on tea leaves [5].Additionally, alongside Fusarium-caused tea root diseases, Poria hypolateritia acts as a fungal pathogen responsible for red root-rot disease, while Phomopsis theae causes Phomopsis (collar) stem canker disease, both posing significant concerns that impact the growth, yield, and quality of tea plants [8,9].These diseases can be severely detrimental to tea production, resulting in notable losses in both yield and quality for tea growers.Therefore, effective monitoring and control measures must be implemented to manage Fusarium diseases on tea plants.
During a survey of tea plantations in Zhejiang, we observed that some tea trees exhibited signs of wilt, and we collected diseased samples and isolated and identified the pathogens responsible for the wilt of the tea trees.This research focuses on the identification and pathogenicity associated with tea rot.By gaining a deeper understanding of the specific Fusarium species, the tea industry can implement more targeted and effective management strategies.

Sample collection and fungal isolation
Tea plants with rot were collected from Anji, Huzhou and Yuhang, Hangzhou tea plantations.Fungal strains were isolated following the method described by Tang [10].The diseased root and stem samples were surface disinfected with 75% ethanol for 1 min followed by 0.5% sodium hypochlorite solution for 2 min and then soaked in sterile distilled water three times to remove the sterilizing agent.Samples were dried by blotting, and disease tissues were cut into small pieces (3 mm 2 ) and then placed on potato dextrose agar (PDA) (BD Difco) with ampicillin (50 µg/mL) and rifampicin (25 µg/mL) antibiotics.The plates were incubated at 25 °C until hyphae emerged from the tissue.A small amount of fresh hyphal tips were cut and transferred onto PDA and incubated at 25 °C.

DNA extraction, PCR, and sequencing
The isolates were used for DNA extraction and sequencing.For each isolate, the mycelium taken from a 7-dayold culture grown on PDA was frozen and homogenized.Genomic DNA was extracted using the 2% cetyltrimethylammonium bromide (CTAB) method [11].PCR amplification was performed for the internal transcribed spacer region (ITS) of ribosomal DNA, translation elongation factor 1-alpha (tef-1α), beta-tubulin (tub2), and RNA polymerase II second largest subunit (rpb2).The primer pairs used in this study are listed in Table 1.All PCR products were subjected to electrophoresis in 2% agarose gel, purified by a gel extraction kit (Vazyme, Nanjing, China), and sequenced by the dideoxy termination method at Sangon Biotech (Shanghai) Co., Ltd, China.The ITS, tef-1α, tub2 and rpb2 gene sequences were deposited in GenBank (Table 2).

Phylogenetic analyses
The DNA sequences of ITS, tub2, tef-1α, and rpb2 were used in the phylogenetic analyses.Closest matches were identified by BLAST (Basic Local Alignment Search Tool) searches in the National Center for Biotechnology Information (NCBI) database (https://blast.ncbi.nlm.nih.gov/Blasnt.cgi) of sequences available in GenBank.Reference sequences of the F. fujikuroi species complex (FFSC), Fusarium oxysporum species complex (FOSC), and Fusarium solani species complex (FOSC) were downloaded from GenBank following references and are listed in Table 2.The MAFFT v. 6.864b (https://mafft.cbrc.jp/alignment/server/index.html)online tool was used to obtain multiple sequence alignments, which were visually inspected and improved manually when necessary.The tree was generated on MEGA 11 with default parameters and bootstrapping with 1000 replicates to obtain the maximum-likelihood (ML) tree [12].

Gene primer pairs References internal transcribed spacer region (ITS) of ribosomal DNA ITS1: C T T G G T C A T T T A G A G G A A G T A A ITS4: T C C T C C G C T T A T T G A T A T G C [22]
translation elongation factor 1-alpha (tef-1α) EF-1: ATGGGTAAGGARGACAAGAC EF-2: GGARGTACCAGTSATCATGTT [23] beta-tubulin (tub2)

Bt-F: A A C A T G C G T G A G A T T G T A A G T Bt-R: T C T G G A T G T T G T T G G G A A T C C [24]
RNA polymerase II second largest subunit (rpb2) RPB2-5F2: GGGGWGAYCAGAAGAAGGC RPB2-11aR: GCRTGGATCTTRTCRTCSACC [25,26]

Fusarium morphological observations
For morphological characterization, mycelial discs (3 mm in diameter) were taken from the growing edge of 5-dayold cultures in triplicate, inoculated onto fresh plates of PDA and incubated in the dark at 25 °C.After a 5-day incubation period, the shape, color, and density of colonies were recorded.For the morphological characterization of fungal conidia, fungal strains were grown in 100 mL portions of potato dextrose broth (PDB) at 25 °C for 4 days, and the spores were collected by culture filtration using two layers of cheesecloth.Spores were centrifuged and resuspended in ddH 2 O at a concentration of 1 × 10^5 mL − 1 .The shape, color, and size of the conidia were observed using light microscopy (Leica ICC50 W, Germany).The isolates were cultured on mung bean culture medium to observe macroconidia.

Tea plant pathogenicity assay
The Fusarium isolates were tested for pathogenicity following the chaff-grain medium method described by Leslie and Summerell, with modifications [11].Briefly, cereal chaff and grain were mixed together in an approximately 5:1 ratio, and then 200 mL tap water was added and mixed thoroughly to release any air bubbles and placed at 4 °C overnight.The chaff-grain mixture was wrapped in cheesecloth and drained until no more water could be released.The mixture was distributed into Erlenmeyer flasks and autoclaved for 15 min on two successive days.Containers were inoculated with conidial suspensions (10^5 cfu/mL) at a rate of 1 mL suspension per 100 mL chaff-grain mixture.The inoculated material was shaken vigorously and then incubated at 25 °C for 10 days until the medium was completely colonized.The culture was removed from the container and air-dried at room temperature overnight.Pathogenicity tests were conducted on one-year-old tea plants of the Camellia sinensis 'Huangjinya' cultivar.In pots, the inoculum was mixed with disinfected vermiculite at a rate of 2% of the final total volume before planting.The inoculated tea tree pots were placed under field conditions for tea plants, and each treatment was watered in a standardized manner.The status of the tea trees was then observed and recorded.The pathogen was reisolated from the tree after the development of symptoms.

Disease sample symptoms
During the autumn of 2020, tea bushes in the tea plantations of Anji, Huzhou (119.87970E, 30.79132N) and Yuhang, Hangzhou (119.4613E, 30.2916N), situated in Zhejiang Province, China, exhibited a range of diseased symptoms with 9% and 15% incidence rates from 'Huangjinya' cultivar.These symptoms were characterized by a reduction in leaf growth, yellowing of leaves, cankers exhibiting visible cracks, peeling of bark, progressive dieback of branches, and eventual death of the entire tree (Fig. 1).The cross-section of the infected stems showed that the vascular bundles turned gray and brown, while healthy plants had lighter colored vascular bundles.The severity of these symptoms suggests the presence of a potential pathogen, prompting the need for further investigation and intervention to prevent the spread of the disease.

Isolation of causal agents
We isolated fungal strains from 10 tea plants showing symptoms of wilt and rot disease.A total of 73 fungal isolates were obtained from the roots and stems of diseased plants.These isolates were subjected to molecular identification using the ITS gene sequences by BLAST searches in the NCBI database.15 isolates of Fusarium, 6 isolates of Botryosphaeria, and 52 isolates of Trichoderma, Penicillium, Mortierella, Poitrasia, and Neurospora were identified.Unexpectedly, for a tea plant that had been dead for a considerable period, we were able to isolate multiple strains of Fusarium from the woody tissues of the tea plants (Fig. 1F-H).To further identify these isolates at the species level, a random selection of eight isolates was subjected to additional analysis.

Identification by DNA barcoding and phylogenetic analysis
Fusarium isolates were subjected to further molecular identification using tub2, tef-1α, and rpb2 gene sequences.The isolates were identified as F. oxysporum (8 isolates), F. fujikuroi (2 isolates), F. solani (4 isolates) and Fusarium concentricum (1 isolate).The sequences derived from the eight fungal isolates obtained in this investigation were submitted to the GenBank database (Table 2).Based on the BLAST results and multigene phylogenetic analyses, strains OS3 and OS4 were identified as F. fujikuroi, and FS7 was identified as Fusarium concentricum, belonging to the F. fujikuroi species complex (FFSC).Fungal isolates FS5, bj2 and FS4 were identified as F. oxysporum belonging to the F. oxysporum species complex (FOSC).Additionally, strains aj2 and ej2 belonged to the F. solani species complex (FSSC).The phylogenetic tree was constructed in this study (Fig. 3), and the results of the phylogenetic analyses revealed topological patterns across the tree with strong bootstrap support for the identification of the isolates.

Pathogenicity of some isolates confirmed by Koch's postulates
The disease symptoms on tea inoculated with the Fusarium isolates are shown in Fig. 4. Eight Fusarium isolates were tested for pathogenicity following the chaff-grain medium method.Each treatment group comprised three pots, with each pot housing three one-year-old tea seedlings, totaling nine seedlings per group.The experiments were conducted independently and repeated three times.Throughout the 30-day duration of each experiment, careful observations were made, and photographs were taken to document the condition of the tea plants.For the control group, the tea seedlings remained healthy throughout the entire period.For the F. fujikuroi OS3 and OS4 strains, the tea seedlings exhibited robust growth during the first six days.However, on the ninth day, some of the leaves began to turn brown, and the tender leaves showed signs of wilting.By the 13th day after inoculation, the OS3 (6 out of 9)-and OS4 (7 out of 9)-inoculated plants showed moderate infections, characterized by rot symptoms.After 30 days after inoculation, the plants displayed severe infections (8 out of 9).The F. oxysporum bj2 strain-inoculated plants displayed three deaths after 30 days of inoculation.The F. oxysporum FS5 strain only resulted in the mortality of one tea seedling.In contrast, no disease symptoms were observed on tea plants treated with FS7, FS4, aj2, and ej2 (Fig. 4).Each fungal isolate was consistently re-isolated from inoculated tissues and reidentified using sequencing methods of characterization to fulfill Koch's postulates.The OS3 and OS4 strains are the same species belonging to F. fujikuroi, and both can cause tea wilt disease in tea plants.For the dead seedlings which were inoculated with either bj2 or FS5, the attempts to re-isolate the strains from these infected plants were not successful.To our knowledge, this is the first report of F. fujikuroi pathogenic fungus in tea plants.

Discussion
This study was dedicated to identifying and comprehending the pathogenicity of fungal diseases responsible for tea rot.The results revealed that the F. fujikuroi isolates were the most aggressive in causing tea rot.We also isolated a number of other strains of Botryosphaeria dothidea, and strains from genera of Penicillium, Mortierella and Trichoderma.These isolates should be analyzed in future studies to test their pathogenicity to tea.Some Trichoderma were isolated from the fine and main roots of a dead tea tree.The isolated Trichoderma may have potential for biological control on the F. fujikuroi OS3 and OS4 disease.Previous research has extensively investigated the potential of Trichoderma in mitigating Fusarium wilt disease in tomato, soybean and banana [13][14][15].The F. fujikuroi OS3 isolates were confirmed based on morphological characteristics and molecular identification.As shown in Fig. 2, the macroconidia of OS3 showed between 3 and 4 septa, and we did not find any macroconidia with five septa.The lack of observation of the macroconidia with five septa may be related to particular media components and cultures older than those in our study, and these older cultures may induce the macroconidia with five septa [16].F. fujikuroi was previously reported to be associated with rice bakanae disease [17], soybean root rot [18], cotton seedling wilt [19], and root rot of tobacco [20].This broad range of host associations underscores the adaptability and versatility of F. fujikuroi as a pathogen.Its presence in various agricultural systems highlights its capacity to exploit diverse plant hosts and potentially cause severe economic losses.Pine-and grass-associated species of the F. fujikuroi species complex were found to have two sets of genes which showed large differences in their ancestral origins, and they tended to occur in sub-telomeric regions of chromosomes underpinning the capacity of these fungi to colonize their respective plant hosts [21].Future studies could test whether the tea infecting F. fujikuroi isolates from our study cause disease in grasses such as rice, and could use a comparative genomics approach to explore the molecular basis of the plant-fungus interactions.It is worth noting that this is the first report of tea rot caused by F. fujikuroi worldwide, indicating the importance of further research and monitoring for this emerging pathogen.
In conclusion, this study sheds light on the challenges posed by fungal diseases in tea production and highlights the importance of comprehensive pathogenicity assessments and identification of emerging pathogens.The discovery of Fusarium strains pathogenic to tea tree could lead to a promising avenue for managing tea plant wilt disease.

Fig. 1
Fig. 1 Symptoms of wilt diseases observed on tea trees in the plantation.(A-B) Wilt diseases caused death of the entire tea plant.(C-D) Wilt caused some branches to show wilt symptoms.(E) Root system browning and decay.(F) Infected plant of tea shown wilt.(G-H) Enlarged image of the roots and stems of the tea plant in F

Fig. 3 Fig. 4
Fig.3Evolutionary analysis by the maximum likelihood method.The percentage of trees in which the associated taxa clustered together is shown below the branches.The bootstrap consensus tree inferred from 1000 replicates is taken to represent the evolutionary history of the taxa analyzed.Evolutionary analyses were conducted in MEGA11.The tree of the Fusarium spp.analyses by ITS, tef-1α, tub2, and rpb2 sequence data.The tree is rooted to Trichoderma harzianum strain CBS 226.95

Table 2
Details of the Fusarium spp.species sequences used in the molecular phylogenetic analysis